BACKGROUND OF THE INVENTION
(a) FIELD OF THE INVENTION
[0001] This invention relates to a process for producing L-threonine by fermentation.
(b) PRIOR ART
[0002] There hitherto has been known that the microorganism belonging to the genus Proteus
or Providencia of which the mutant requires L-isoleucine, can be used as microorganisms
capable of producing L-threonine by fermentation (Japanese Examined Patent Publication
No. 4440/1968).
[0003] However, there is a room for further improvement in the capability of the strains
as to the amount of L-threonine accumulated and as to the yield of L-threonine from
the starting material such as glucose or fructose.
SUMMARY OF THE INVENTION
[0004] One object of the present invention is to provide an improved process for producing
L-threonine by fermentation which can give a much higher accumulated amount and yield.
[0005] - Another object of the invention is to provide an improved process using novel mutanized
microorganisms for producing L-threonine.
[0006] These and other objects of the invention will become more apparent in the detailed
description and examples hereinafter.
These objects are attained by
[0007] A process for producing L-threonine by fermentation which comprises the steps of:
(a) culturing an L-threonine producing microorganism belonging to the genus Providencia
until L-threonine is accumulated in a culture broth said microorganism requiring at
least leucine for the growth thereof and
(b) recovering the accumulated L-threonine from the culture broth.
PREFERRED EMBODIMENTS
[0008] The microorganism used in the present invention belongs to the genus Providencia.
The genus is decided according to Bergy's Mannual of Systematic Bacteriology Volume
1 (1984) pages 495 to 496. Moreover, the microorganism used in the invention requires
at least L-leucine for the growth thereof and is capable to produce L-threonine. In
the invention there may be preferably employed microorganisms which further have the
character lacking threonine aldolase and/or the character having a resistance to lysine
analog.
In the invention lysine analog means
[0009]
(i) the substance which can inhibit the growth of the microorganism belonging to the
genus Providencia, such an inhibition being reversed by supplement of lysine, or
(ii) the substance which can repress or inhibit enzyme in the biosynthetic pathway
of L-lysine.
[0010] Preferable examples of the lysine analog are S-aminoethyl-L-cystein, lysine hydroxamate,
4-hydroxylysine, and so on. As for the lysine analog S-aminoethyl-L-cystein is most
preferably used.
[0011] These characters effectively operate the capability of producing L-threonine.
[0012] Moreover, there may be more preferably employed microorganisms which have further
characters selected from the character requiring L-isoleucine for the growth thereof,
the character having a resistance to threonine analog such as α=amino-β-hydroxyvaleric
acid, and the character having a resistance to methionine analog such as ethionine,
adding to at least one character of the character lacking threonine aldolase and character
having a resistance to lysine analog.
[0013] These characters also effectively operate the capability of producing L-threonine.
Therefore, there may be more preferably employed microorganisms which have some or
all characters above mentioned. These characters can be given to the microorganisms
by conventional method.
[0014] In the present invention, auxotrophy, namely requiring the nutrient for the growth
thereof means wide concept and contains leaky type, namely inoamplete defect type,
and further contains the case when auxotrophy is supplied with biosynthetic precursor
of the required nutrient.
[0015] Representative microorganisms useful for the invention are as follows:
(a) Providencia rettgeri NS-140 (FERM BP-1057) This NS-140 strain has a resistance
to a-amino-S-hydroxyvaleric acid and ethionine; and requires L-isoleucine and L-leucine
for- the growth thereof, and was deposited with Fermeritation Research Institute in
Japan on February 12, 1985.
(b) Providencia rettgeri NS133I-69 (FERM BP-1058) This NS133I-69 strain has a resistance
to a-amino-β-hydroxyvaleric acid and ethionine; requires L-isoleucine and L-leucine
for the growth thereof; and lacks threonine aldolase, and was deposited with Fermentation
Research Institute in Japan on February 12, 1985.
(c) Providencia. rettgeri TP4-105-43 (FERM BP- 1050) This TP4-105-43 strain has a
resistance to a-amino-S-hydroxyvaleric acid, L-ethionine, and S-aminoethyl-L-cysteine;
and requires L-isoleucine and L-leucine for the growth thereof, and was deposited
with Fermentation Research Institute in Japan on July 15, 1985.
[0016] The FERM BP numbers are the access number of the Fermentation Research Institute
Agency of Industrial Science and Technology, at No. 1-3, Yatabe-cho, Higashi 1-chome,
Tsukuba-gun, Ibaragi- ken, 305 JAPAN, from which the microorganisms with FERM BP numbers
are available to any party who requests them.
[0017] These threonine producing microorganisms can be derived as a mutant, for example,
from the following parent strains of Proteus rettgeri which is the previous name of
Providencia rettgeri;
(i) Providencia rettgeri TY-1 (FERM P-8079) This TY-1 strain has a resistance to a-amino-S-hydroxyvaleric
acid and ethionine; and requires L-isoleucine for the growth thereof and is a parent
strain of NS-140.
(ii) Providencia rettgeri NS-133 (FERM P-8079) This NS-133 strain has a resistance
to α-amino-β-hydroxyvaleric acid and ethionine; requires L-isoleucine for the growth
thereof; and lacks threonine aldolase, and is a parent strain of NS133I-69. Moreover,
NS133I-69 strain is a parent strain of TP3-105 which is a parent strain of TP4-105-43.
[0018] In the invention, methods for inducing the mutants are conventional methods such
as irradiation with ultraviolet light, or treatment with N-methyl-N'-nitro-N-nitrosoguanidine
or ethylmethane sulfonate, etc. After the irradiation or treatment, the mutagenized
cells are spread on the minimal agar plates containing a small amount of casamino
acid (Trade Mark) or yeast extract and cultivated at 30 °C for 3 to 5 days. Smaller
colonies formed on the agar plates are isolated, and the colony which requires L-leucine
for the growth thereof is selected.
[0019] When the microorganisms having a resistance to lysine analog is used, we define the
microorganisms having a resistance to lysine analog for the present invention as a
strain which can grow and form colonies on a minimal medium supplemented with 2.5
g/1 of lysine analog and of which the growth degree after the cultivation for 24 hours
is at least 50 %, based on the case in the absence of lysine analog. In the invention
growth degree is shown by the relative optical density of. the culture broth at 660
nm when the optical density of culture broth in none-supplement of lysine analog is
defined as 100 %.
[0020] The process for producing L-threonine using the microorganisms are conventional,
and the microorganisms are cultivated in a conventional medium containing carbon sources,
nitrogen sources, inorganic salts and other necessary organic minor nutrients.
[0021] There can be used as a carbon source, carbohydrates such as glucose, fructose, starch,
cellulose hydrolysate, or molasses; organic acid such as fumalic acid, citric acid,
or succinic acid; alcohol such as glycerol.
[0022] There can be used as a nitrogen source, organic ammonium compound such as ammonium
acetate, or urea; inorganic ammonium compound such as ammonium sulfate, ammonium chloride,
ammonium phosphate, or ammonimum nitrate; ammonia gas; aqueous ammonia.
[0023] There can be preferably used as organic nutrient corn steep.liquor, polypeptone,
or yeast extract.
[0024] There can be used as a inorganic salt, potassium phosphate, magnesium sulfate, ferrous
sulfate 7-hydrate or 4-to 6-hydrate of manganese sulfate.
[0025] The preferable culture medium may contain 2 to 15 % of carbon source, 0.5 to 4.0
% of nitrogen source, and 0.001 to 0.4 % of required organic material, 0 to 4 % of
natural organic nutrient and minor amount of inorganic nutrient.
[0026] Cultivation is carried out under aerobic conditions such as being shaken or stirred
with aeration at a temperature from 24 ° to 37 °C for 48 to 120 hours. During cultivation
the pH of the medium is adjusted to 5 to 9.
[0027] After cultivation, L-threonine in the culture broth thus obtained can be separated
by a known method, for example, by means of ion-exchange resins. In order to recover
the accumulated L-threonine from the culture broth, the microorganisms are removed
off from the culture broth with centrifuging, and the resulting culture broth solution
is adjusted to pH 2 by hydrochloric acid, and then the broth 'solution is passed through
the strongly acidic cation exchange resin. Thereafter, the adsorbant is eluted by
dilute aqueous ammonia. Ammonia is evaporated from the resulting eluent, and then
the resulting solution is condensed. The alcohol is added to the resultant and left
standing under cooling to give crystals of L-threonine.
[0028] The invention will be more clearly understood with reference to the following Experiments
and Examples. However, these Experiments and Examples are intended to illustrate the
invention and are not to be construed as limiting the scope of the invention.
EXPERIMENT 1
[0029] Isolation of the mutant strain requiring L-leucine for the growth thereof
[0030] Providencia rettgeri TY-1 and NS-133 were irradiated with ultraviolet light by a
conventional method, respectively. The mutagenized cells were spread on the agar plates
shown in Table 1.
![](https://data.epo.org/publication-server/image?imagePath=1987/01/DOC/EPNWA2/EP86106204NWA2/imgb0001)
[0031] Then, the agar plates were incubated for 4 to 6 days at 30 °C. In the colonies formed
on the plate, smaller colonies were picked up from the colonies formed on the plate.
NS-140 strain, which has a resistance to a α-amino-β-hydroxyvaleric acid and ethionine
and requires L-isoleucine and L-leucine, was obtained from the parent TY-1 strain.
[0032] NS133I-69 strain, which has a resistance to a-amino -β-hydroxyvaleric acid and ethionine,
requires L-isoleucine and L-leucine for the growth thereof, and lacks threonine aldolase,
was obtained from the parent NS-133 strain.
EXPERIMENT 2
[0033] Isolation of the mutant strain requiring L-leucine for the growth thereof and having
a resistance to S-aminoethyl-L-cysteine
Providencia rettgeri NS133I-69 strain was irradiated with ultraviolet light by a similar
manner to Experiment 1. TP3-10-5 strain, which has a resistance to α-amino-β-hydroxyvaleric acid and L-ethionine,
and requires L-isoleucine and L-leucine, was obtained: TP3-105 strain was treated
with N-methyl-N'-nitro-N-nitrosoguanidine by a conventional method (300 µg/mℓ,30 °C,
10 minutes).
[0034] The mutagenized cells were spread on the agar plate shown in Table 2.
![](https://data.epo.org/publication-server/image?imagePath=1987/01/DOC/EPNWA2/EP86106204NWA2/imgb0002)
[0035] Then the agar plates were incubated for 5 to 7 days at 30 °C. In the colonies formed
on the plate, larger colony was picked up from colonies formed on the plate and obtained
TP4-105-43 strain which has a resistance to α-amino-β-hydroxyvaleric acid, L-ethionine
and S-aminoethyl-L-cysteine and requires L-isoleucine and L-leucine for the growth
thereof.
EXPERIMENT 3
Test of L-leucine auxotrophy
[0036] The each strain show in Table 4 was cultivated on nutrient agar slant for 24 hours:
The resulting strains were placed on the minimal agar plate shown in Table 3 containing
no L-leucine and 0.01 % of L-leucine and cultivated at 30 °C for 4 days.
![](https://data.epo.org/publication-server/image?imagePath=1987/01/DOC/EPNWA2/EP86106204NWA2/imgb0003)
[0037] The growth degree was measured. The mutant requiring L-leucine was determined by
one which is incapable or difficult to grow in the absence of L-leucine and capable
to grow in the presence of L-leucine.
[0038] The results are shown in Table 4.
[0039] Providencia rettgeri NS-140, NS1331-69, and TP4-105-43 apparently require L-leucine
for the growth thereof, while Providencia rettgeri TY-1 and NS-133 which are parent
strain, do not require.
![](https://data.epo.org/publication-server/image?imagePath=1987/01/DOC/EPNWA2/EP86106204NWA2/imgb0004)
EXPERIMENT 4
Test of resistance to S-aminoethyl-L-cystein
[0040] Each microorganism shown in Table 6 was cultivated in vouillon liquid at 30 °C for
16 hours with shaking, was harvested, and washed well with physiological saline. The
resulting cell suspension of each microorganism were inoculated into 5 ml of the minimal
medium shown in Table 5 containing 0 g/ℓ, 2.5 g/ℓ, 5 g/ℓ, 7.5 g/ℓ, 10 g/ℓ, respecitively,
and cultivated at 30 °C for 14 hours.
![](https://data.epo.org/publication-server/image?imagePath=1987/01/DOC/EPNWA2/EP86106204NWA2/imgb0005)
[0041] The growth degree was measured.
[0042] The results are shown in Table 6. Providencia rettgeri TP4-105-43 is not inhibited
the growth in the presence of the high concentration of S-aminoethyl-L-cysteine and
have a strong resistance to S-aminoethyl-L-cysteine.
![](https://data.epo.org/publication-server/image?imagePath=1987/01/DOC/EPNWA2/EP86106204NWA2/imgb0006)
EXAMPLE 1
Production of L-threonine
[0043] Each microorganism shown in Table 8 was cultivated in vouillon liqiud at 30 °C for
16 hours with shaking to give seed culture broth. Then, 4 ml of seed culture broth
were inoculated into 40 ml of the fermentation medium shonw in Table 7 in 1-liter
shaking flask. Cultivation was carried out at 30 °C for 90 hours with shaking conditions(150
rpm, 3 cm stroke).
![](https://data.epo.org/publication-server/image?imagePath=1987/01/DOC/EPNWA2/EP86106204NWA2/imgb0008)
[0044] After cultivation, the medium was filtrated and removed off microorganisms and calcium
carbonate, the amount of L-threonine accumulated in the resulting filtrate was quantitative
analyzed by automatic amino acid analyser (Produced by Japan Electric Co. JLC 200A).
The results are shown in Table 8.
![](https://data.epo.org/publication-server/image?imagePath=1987/01/DOC/EPNWA2/EP86106204NWA2/imgb0009)
[0045] In examples of the present invention, the amount of L-threonine accumulated and the
yield were efficiently improved, compared with the parent strains.
EXAMPLE 2
Production of L-threonine
[0046] Providencia rettgeri NS-140 was cultivated in vouillon liquid medium at 30 °C for
16 hours with shaking to give seed culture broth. 100 ml of the seed culture was transferred
into the 2-liter jar fermentor, which contained 900 ml of the same fermentation- medium
as used in Example 1 except that 0.5 % of (NH
4)
2SO
4 and 4.0 % of glucose were used. Cultivation was carried out at 30 °C with agitation
(800 rpm) and with aeration (1 liter of air per min). PH was controlled to 6.5 to
8.0 by 25 % aqueous ammonia which was used as a nitrogen source. During cultivation,
glucose was fed intermitlently and 150 g of glucose were consumed.
[0047] After cultivation of 76 hours, 26.0 g/1 of L-threonine, which was 17.3 wt% based
on glucose, was produced.
[0048] The culture broth was centrifuged and the microorganisms were removed off from the
culture broth. 500 ml of the filtrate was passed through the column packed with supernatant
strong cation exchange resin DIAION (Trade Mark) SK·lB [H type]. Then, column was
washed by water and thereafter the adsorbant in column was eluted by 2 N aqueous ammonia.
The eluent was decolorized and condensed under reduced pressure. The alcohol was added
to the resultant and left standing under cooling to give crystals of L-threonine.
The crystals were gathered, dried to give 11.5 g of L-threonine having 96 % of the
purity.
1. A process for producing L-threonine by fermentation which comprises the steps of:
(a) culturing an L-threonine producing microorganism belonging to the genus Providencia
until L-threonine is accumulated in a culture broth said microorganism requiring at
least leucine for the growth thereof and
(b) recovering the accumulated L-threonine from the culture broths
2. A process accoding to claim 1, wherein said microorganism further requires isoleucine
for the growth thereof and has a resistance to α-amino-β-hydroxyvaleric acid and ethionine.
3. A process according to claim 1, wherein said microorganism has a resistance to
a lysine analog.
4. A process according to claim 1, wherein said microorganism further requires isolcucine
for the growth thereof, and has a resistance to a lysine analog, α-amino-β-hydroxyvaleric
acid and ethionine.
5. A process according to claim 1, wherein said microoganism lacks threonine aldolase.
6. A process according to claim 1, wherein said microorganism further requires isoleucine
for the growth thereof, has a resistance to a-amino-P-hydroxyvaleric acid and ethionine,
and lacks threonine aldolase.
7. A process according to claim 1, wherein said microorganism has a resistance to
a lysine analog, and lacks threonine aldolase.
8. A process according to claim 3, 4, or 7, wherein lysine analog is S-aminoethyl-L-cysteine.